TMB Peroxidase Substrate Solution Blue

Tony Rook

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Nov 16, 2015
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Please find the following media preparation method:

TMB Peroxidase Substrate Solution Blue


Final Dilution:

0.005% TMB - 0.006% H2O2 in 0.01M Acetate Buffer and
0.05% Sodium Nitroprusside toxic (or Sodium
Nitroferricyanide less toxic), pH3.3



Stock Solutions:


0.1% TMB (20x) in 100% Ethanol:

Add 0.01 g of TMB (3,3, 5,5 tetramethyl benzidine, Sigma)
in 10 ml of 100% ethanol (may be warmed to 37-40 °C to
dissolve TMB) and store at 4 °C.


0.3% H2O2 (50x) in Distilled Water:

Add 100 ul of H2O2 in 10 ml of distilled water and store at
4 °C.


0.2M Acetate Buffer-1% Sodium Nitroferricyanide (20x), pH3.3

Sodium acetate, trihydrate (MW 136.1) --------- 2.72 g

Sodium nitroferricyanide ------------------------ 1 g

Distilled water --------------------------------- 100 ml

Mix to dissolve and adjust pH to 3.3 using concentrated HCl
and store at 4 °C.


0.01M Acetate Buffer - 0.05% Sodium Nitroferricyanide, pH 3.3:

0.2M Acetate buffer - 1% sodium nitroferricyanide --- 1 ml

Distilled water ------------------------------------- 19 ml

Mix well and adjust pH if necessary.


Working Solution:

Add 5 drops of 0.1% TMB to 5 ml of 0.01M acetate buffer
0.05% sodium nitroferricyanide.

Add 2 drops of 0.3% H2O2 and mix well.

Incubate sections for 20-30 minutes at room temperature.


Note: This is very sensitive solution and will produce more background staining than other peroxadase substrate solutions.


Link Reference:
http://www.ihcworld.com/_protocols/chromogen_substrates/POD_TMB_blue.htm